Pseudolaric acid-B derivatives, their preparation and pharmaceutical compositions

ABSTRACT

The invention relates to pseudolaric acid-B derivatives of general formula (I), wherein (a) R 1  is cyano, heterocyclyl, COXR′ or CON(R″) 2 , wherein X is O or NH, R′ is H, cycloalkyl, alkyl, heterocyclic alkyl or arylalkyl, each R″ is independently alkyl, cycloalkyl or heterocyclicalkyl; (b) R 2  is H, alkylacyl, arylalkylacyl, arylacyl or heterocyclylacyl; (c) R 3  is COXY, amino or halogen, wherein X is O or NH, Y is H, NH 2 , hydroxy, alkyl, cycloalkyl, heterocyclicalkyl, hetroatom-substituted alkyl, tertiary amino-substituted ammonioalkyl, aryl, arylalkyl or polyhydroxyalkyl. The invention also relates to processes for preparing such derivatives and antitumor or antifungal pharmaceutical compositions containing the same.

TECHNICAL FIELD

The invention relates to semi-synthetic antitumor or antifungalmedicines, especially relates to novel semi-synthetic pseudolaric acidderivatives, the processes for preparing such derivatives and antitumoror antifungal pharmaceutical compositions containing the same.

BACKGROUND OF THE INVENTION

It has been found in the recent investigations that pseudolaric acidsand the compounds with correlative structures possess importantbiological activities. The methods for the isolation of such compoundsand the pharmacologically experimental results have been disclosed inthe following references:

1. Wu Saoxi, et al., Chinese Journal of Dermatology, 1960 (8): 18;

2. Li Zhulian, Zhou Bingnan, et al., “The proceeding of sino-Americansymposium on chemistry of Natural products”, <<Chemistry of NaturalProducts>> (1982), pp.150-155;

3. Li Zhulian, Xu Guangyi, et al., “Study of new diterpenoids frompseudolarix kaempferi”, Acta Chimica Sinica, 1982 (40): 447-456;

4. Wang Weichen, et al., “Action of anti-procreation of pseudolaricacid-B”, Acta Pharmacol. Sinica, 1982 (3): 188-192;

5. Erguang Li, Alice M. Clark and Charles D. Hufford, “Antifugalevaluation of Pseudolaric acid B, a major constituent of pseudolarixkaempferi”, Journal of Natural Product, 1995 (58): 57-67;

6. De-Ji Pan, Zhu-lian Li, et al., “The cytotoxic principles ofpseudolarix kaempferi: Pseudolaric acid-A and -B and relatedderivatives”, Plant Medica, 1990 (56): 383-385.

All the literatures mentioned above are incorporated herewith forreference.

It was reported that the angiogenesis in the tumor tissues was relatedwith the malignant proliferation and metastasis of the tumor. Theinhibition of tumor angiogenesis will lead to the decrease of suppliesof oxygen and nutrition which were necessary for the tumor growth, thenthe tumor proliferation will be inhibited. Meantime, anti-angiogenesisalso prevents the tumor metastasis because it can block the pathwaythrough which tumor cells can enter into the blood circulation fromtumor tissue via neonate capillaries.

The literatures in respect to the relationship between angiogenesis andcancers were listed as follows:

1. Cockerill G. W., Gamble T. R., Vadas M. A., “Angiogenesis: models andmodulators”, Int. Rev. Cytol., Vol.159(1995), pp.113-160;

2. Senger D. R., Van de water L., Brown L. F., et al., “Vascularpermeability factor(VPF, VEGF) in tumor biology”, Cancer MetastasisRev., Vol.12(1993), pp.303-324;

3. Donovan D., Harmey J. H., Redmond H. P. et al., “Ascites revisited: anovel role for tamoxifen”, Eur. J. Surg. Oncol., Vo.123(1997), pp. 570;

4. Ingber D, Fujita T., Kishimoto S. et al., “Synthetic analogues offumagillin that inhabit angiogenesis and suppress tumor growth”, Nature,Vol.348 (1990), pp. 555-560;

5. Jiang W. G., Puntis M. C. A., Hallett M. B., “molecular and cellularbasis of cancer invasion and metastasis: implications for treatment”,Br. J. Surg., Vol.81 (1994), pp. 1576-1590;

6. Fidle I. J., Gerstein D. M., Hart I. R., “The biology of cancerinvasion and metastasis”, Adv. Cancer Res., Vol.28(1978), pp.149-250;

7. Hanahan D., Folkman J., “Patterns and emerging mechanisms of theangiogenic switch during tumorigenesis”, Cell., Vol.86(1996), pp.353-364;

8. Ferrara N., “The role of vascular endothelial growth factor inpathological angiogenesis”, Breast Cancer Res. Treat, Vol.36(1995), pp.127-137;

9. O'Reilly M., Holmgren L., Shing Y., et al., “Angiostain: anovelangenesis inhabitor that medicates the suppression of metastases by aLewis lung carcinoma”, Cell., Vol.79 (1994), pp. 689-692;

10. O'Reilly M. S., Holmgren L., Chen C., et al., “Angiostatin inducesand sustains domancy of human primary tumors in mice”, Nature Med.,Vol.2 (1996), pp.689-692;

11. O'Reilly M. S., Boehm J., Shing Y., et al., “Endosatatin: anendogenous inhabitator of angiogenesis and tumor growth”, Cell., Vol.88(1997), pp. 277-285;

12. Frank R. E., Saclarides T. J., Sue Leurgans, et al., “Tumorangiogenesis as a predictor of recurrence and survival in patients withnode-negative colon cancer”, Ann. Surg., Vol.222 (1995), pp. 695-699.

All the literatures mentioned above are incorporated herewith forreference.

Although it was known that the use of angiogenesis inhibitors in thecancer chemotherapy could increase therapeutic effects and prevent themetastasis and relapse of the tumor, but no new drug targeting atangiogenesis has been marketed nowadays.

During long-time researches, the inventors have found that pseudolaricacids and new derivatives thereof could inhibit the growth of humanmicrovascular endothelial cells, thus they could decrease supplies ofoxygen and nutrition, which were necessary for tumor cells, byinhibiting the formation of blood vessels in the tumor tissue. Thereby,the cleavage and proliferation of tumor cells would be inhibited,resulting in the atrophy and vanishing of the tumor.

Accordingly, one purpose of the invention is to provide novelpseudolaric acid derivatives.

Another purpose of the invention is to provide a process for preparingsaid derivatives.

A further purpose of the invention is to provide antitumor or antifungalpharmaceutical compositions containing the same.

SUMMARY OF THE INVENTION

The invention provides pseudolaric acid derivatives having the followingformula (I):

wherein

(a) R₁ is a cyano, a heterocyclic group, COXR′ or CON(R″)₂, wherein X isO or NH, R′ is H, a cycloalkyl, an alkyl, a heterocyclic alkyl or anarylalkyl, each R″ is independently an alkyl, a cycloalkyl or aheterocyclic alkyl;

(b) R₂ is H, an alkylacyl, an arylalkylacyl, an arylacyl or aheterocyclic acyl; and

(c) R₃ is COXY, an amino or a halogen, wherein X is O or NH, Y is H,NH₂, a hydroxy, an alkyl, a cycloalkyl, a heterocyclic alkyl, an alkylsubstituted by oxygen(s), an aryl, an arylalkyl or a polyhydroxyalkyl,

Provided that pseudolaric acid A, pseudolaric acid B, pseudolaric acidC, pseudolaric acid C₂, deacetoxyl pseudolaric acid A, deacetoxylpseudolaric acid C₂, methyl pseudolarate A, deacetoxyl methylpseudolarate A, p-bromine phenacyl pseudolarate A, methyl pseudolarateB, p-bromine phenacyl pseudolarate B, methyl pseudolarate C, propylpseudolate C, iso-propyl pseudolate C, 19-demethoxyl iso-propylpseudolarate B, 19-demethoxyl iso-propyl pseudolarate C, 19-propyoxylpseudolarate B, 19-propoxyl pseudolaric acid C, 19-butoxyl pseudolaricacid B, and 19-butoxyl pseudolaric acid C are excluded.

The invention also provides the following processes for preparingpseudolaric acid derivatives having formula (I):

a. In case R₁ is CONHR or CONR′R″, aminolyzing a compound of formula (I)wherein R₁ is COOR′ in aqueous amine (said compound:saidamine=1:1˜1:300) in the presence of an acid catalyst at the conditionsof temperature from −10° C. to 100° C. and pH 1-6;

b. In case R₁ is COOR′, alcoholyzing a compound of formula (I) whereinR₁ is COOR′ with an excess alcohol (said compound:saidalcohol=1:1˜1:500) in the presence of an alkaline catalyst underanhydrous condition at a temperature from 0° C. to the refluxtemperature of the solvent and pH 9-14;

c. In case R₂ is alkylacyl, aryl-substituted alkylacyl, arylacyl orheterocyclylacyl, acylating a compound of formula (I) wherein R₂ is H(said compound:acylating agent=1:5˜1:500) at a temperature from 0° C. to80° C.; or

d. In case R₃ is COXY, reacting a compound of formula (I) whereinR₃=COOH with an excess acyl halogenating agent to form an acyl halide,then reacting said acyl halide with an alcohol or an amine in thepresence of an acid scavenger at a temperature between −20˜30° C.

The invention further provides a pharmaceutical composition for thetreatment of tumors or inhibition of fungi, comprising a therapeuticallyeffective amount of a pseudolaric acid-B derivative of formula (I) and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

Unless indicated otherwise, the substituents used herein have thefollowing definitions: “alkyl” means a substituted or unsubstituted,saturated or unsaturated linear or branched alkyl chain, preferably,propyl, 2-methylpropyl or 2,2-dimethylpropyl; “cycloalkyl” means asubstituted or unsubstituted 5- or 6-membered cycloalkyl, preferably,cyclohexyl; “aryl” means a substituted or unsubstituted, heterocyclic ornonheterocyclic aryl group; “arylalkyl” means an alkyl with substitutedor unsubstituted aryl(s) thereon; “heterocyclic group” means a 5- or6-membered heterocyclic group; “heteroalkyl” means a substituted orunsubstituted aromatic or non-aromatic heterocyclic alkyl, preferably,(α-furyl)methylene (furfural).

The preferred pseudolaric acid-B derivatives of the invention are thosewherein R₁ is CONHR′ or CON(R″)₂, R₂ is H, an alkylacyl, anarylalkylacyl, an arylacyl, or a heterocyclic acyl, and R₃ is —COOH.

The preferred pseudolaric acid-B derivatives of the invention are alsothose wherein R₁ is —COOR′, R₂ is H, an alkylacyl, an arylalkylacyl, anarylacyl, or a heterocyclic acyl, and R₃is —COOH.

The preferred pseudolaric acid-B derivatives of the invention are alsothose wherein R₁ is —COOR′, R₂ is H, an alkylacyl, an arylalkylacyl, anarylacyl, or a heterocyclic acyl, and R₃ is COXY.

In the substituents of the compounds according to the invention,arranged by activities, preferred R₁ is an ester alkyl, preferred R₂ isan acetyl and preferred R₃ is a carboxyl or a (m-hydroxyanilino)acyl.

The compounds of the invention can be obtained via a several-stepreaction by using pseudolaric acid-B or compounds having the stemnucleus of pseudolaric acid as a starting material, the preparationthereof was shown in De-Ji Pan, Zhu-lian Li, et al., “The cytotoxicprinciples of pseudolarix kaempferi: Pseudolaric acid-A and -B andrelated derivatives”, Plant medica, 1990 (56): 383-385. The process forpreparing the compounds of the invention is superior in mild reaction,simple method, high yield and easy industrialization.

The compounds of the invention can be prepared by adopting one of thefollowing schemas or the combination of some of them.

wherein, each of R_(m) and R′_(m), same or different, can independentlybe H, methyl, ethyl, propyl, pentyl or hexyl, orR_(m)=R′_(m)=—(CH₂)_(n)—, wherein n=2-5.

The acid catalyst can be selected from a group consisting of H₂SO₄,H₃PO₄, CH₃CO₂H and HCl, preferrably, CH₃CO₂H.

The reaction can be conducted with stirring in water or an organicsolvent. The organic solvent can be a liquid amine itself or benzene,chloroform, ether, tetrahydrofuran, etc. The temperature of the reactionis −10˜100° C.

The alkaline catalyst can be selected from metal ammines, eg., sodiumamide and potassium amide; and metal alkoxide, eg., sodium alkoxide andpotassium alkoxide; preferably, sodium alkoxide and potassium alkoxide.

The reaction is conducted by stirring the reaction mixture in ananhydrous solvent, which itself is a reaction reagent or selected fromother inert solvents, eg., anhydrous ether, tetrahydrofuran or tertiarybutanol, etc. The temperature of reaction is from −4° C. to the refluxtemperature of the solvent.

The catalyst in the above reaction can be DCC, AgCN, DMAP or pyridine,preferably, AgCN.

Generally, the acylating agent can be an acyl halide. When actylchloride or propionyl chloride, etc., is used as an acylating agent, thecatalyst can be omitted.

When the solvent is an acyl halide itself or aqueous ether, the reactionis conducted at peripheral temperatures with stirring or in refluxconditions.

The reaction is conducted at peripheral temperatures with stirring or inreflux conditions.

WH is an alcohol, a phenol, a primary amine or a secondary amine.

The acid scavenger can be an organic amine, CaCO₃, K₂CO₃, Na₂CO₃,NaHCO₃, KOH or NaOH, etc., preferably, Na₂CO₃ or NaHCO₃.

The composition according to the invention comprises a compound of theinvention in an amount of safety and within a range of effective dosageand a pharmaceutically acceptable carrier.

“A safe and effective dosage” means that the amount of the compound isenough to obviously ameliorate conditions of diseases while not inducingserious side effects. The safe and effective dosage of the compounddepends on specific conditions, such as the age of the patient, thecondition of disease, the period of treatment, etc. Preferably, thecomposition of the invention includes the compound of the invention inan amount from 0.1% (by weight) to 99.9% (by weight), more preferably,from 20% (by weight) to 60% (by weight).

“The pharmaceutically acceptable carrier” means one or more compatiblesolid or liquid filler(s) or gel material(s) which are suitable to usefor human and have enough purity and low enough toxicity. “Compatible”means, herein, that each of ingredients in the composition can beincorporated each other or with the compound of the invention, while noobvious decrease will be seen in the effect of the compound. Examples ofthe pharmaceutically acceptable carrier are as follows: sugars, eg.,glucose, sucrose and lactose; starches, eg., corn starch and potatostarch; cellulose and derivatives thereof, eg., carboxymethylcellulosesodium, ethylcellulose sodium and cellulose acetate, etc.; gelatin;talc; solid lubricants, eg., stearic acid and magnesium stearate, etc.;calcium sulfate; vegetable oils, eg., soya bean oil, sesame oil, peanutoil and olive oil, etc.; polyols, eg., propylene glycol, glycerin,mannitol and sorbitol, etc.; emulsifiers, eg., Tween®; lubricants, eg.,sodium dodecyl sulfate; colouring agents; flavorings; stabilizers;antioxdants; preservatives; apyrogentity water; etc. The selection onthe carrier depends on the administration manner of the compound. Whenthe composition of the invention is used as an antitumor drug, it ispreferably taken orally or parenterally, more preferably, parenterally.When the composition of the invention is used as an antifungal, it ispreferably taken orally, topically or parenterally.

Preferred Embodiments

A part of the compounds prepared by using the process according to theinvention have specific structures exemplified in Table 1.

TABLE 1 (I)

No. R₁ R₂ R₃ PEX-1 CO₂CH₂CH(CH₃)₂ H CO₂H PEX-4 CO₂CH₂CH₃ H CO₂H PEX-4YCO₂CH₂CH₃ AC CO₂H PEX-6Y CO₂CH₃ AC CONH₂ PEX-7Y CO₂CH₃ AC CONHOH PEX-8YCO₂CH₃ AC CON(CH₂)₂ PEX-9Y CO₂CH₃ AC CONHCH₂CH₂OH PEX-10Y CO₂CH₃ ACCO₂CH₂CH₃ PEX-11Y CO₂CH₃ AC CONHC(CH₂OH)₃ EX-12Y CO₂CH₃ AC CONHCH₂CO₂HPEX-13Y CO₂CH₃ AC CONHCH₂CONH₂ PEX-14Y CO₂CH₃ AC COph PEX-15Y CO₂CH₃ ACCONH-(m-OH)ph PEX-16Y CO₂CH(CH₃)₂ AC CO₂H PEX-18Y CO₂CH₂ph AC CO₂HPEX-21 CO₂(CH₂)₂CH(CH₃)₂ H CO₂H PEX-21Y CO₂(CH₂)₂CH(CH₃)₂ AC CO₂HPEX-23Y CO₂CH₃ COph CO₂H PEX-24Y CO₂CH₃ COCH₂(P-Cl)ph CO₂H PEX-25YCO₂CH₂(α-furyl) AC CO₂H PEX-26Y CO₂CH₃ CO(α-furyl) CO₂H PEX-30YCO₂CH₂(α-tetrafuryl) AC CO₂H PEX-30 CO₂CH₂(α-tetrafuryl) H CO₂H PEX-31YCO₂CH₂(cycloheptayl) AC CO₂H PAM-1Y CONH₂ AC CO₂H PAM-2Y CONH₂ H CO₂HPAM-5Y CONHCH₃ H CO₂H PAM-7Y CON(CH₂CH₂)₂ H CO₂H PAM-8Y CONHCH₃ AC CO₂HPBX-1Y CO₂CH₂CH(CH₃)₂ AC CO₂H PBX-2Y CO₂CH₂C(CH₃)₃ AC CO₂H PBX-3Y CO₂MeCO(CH₂)₂CH₃ CO₂H PBX-4Y CO₂(CH)₂OH AC CO₂H PBX-5Y 2-oxazoline AC CO₂HPBX-6Y CO₂CH₂CH(C₂H₅)₂ AC CO₂H PBX-7Y CO₂CH₂CH(SCH₂)₂CH₂ AC CO₂H PBX-8YCO₂CH₂CH(CH₂CH₂)₂ AC CO₂H PBX-9Y CO₂(CH₂)₂CH(CH₃)₂ AC CO₂H PBX-10YCO₂CH(CH₂)₂ AC CO₂H PBX-11Y CO₂(CH₂)₃OCH₃ AC CO₂H PBX-12Y CO₂(CH₂)₂OCH₃AC CO₂H PBX-13Y CO₂CH₂CH₂NH₂ AC CO₂H PBX-14Y CO₂CH₂CH₂SCH₃ AC CO₂HPBX-15Y CO₂CH₂CH₂CN AC CO₂H PBX-16Y CO₂CH₂CF₃ AC CO₂H PBX-17YCO₂CH₂CH(SCH₂)₂ AC CO₂H

TABLE 2 MS and IR data of some compounds of the invention ESI-MSIonization Deprotonated No. type ion peak IR (cm⁻¹), KBr disc PEX-1 +455(M + 23) — PEX-4Y + 469(M + 23) 3434.7, 1741.4, 1706.7, 1643.1PEX-4 + 427(M + 23) — PEX-6Y + 454(M + 23) 3500, 3392.2, 1739.5, 1700,1646.9 PEX-7Y + 470(M + 1) 3545, 3367.2, 1739.5, 1701, 1648.9 PEX-8Y +458(M + 1) 3428.9, 1739.5, 1708.6, 1643.1 PEX-9Y + 476(M + 1) 3407.7,1739.5, 1710.6, 1648.9 PEX-10Y + 483(M + 23) 3453.9, 1741.4, 1708.6,1643.1 PEX-11Y + 536(M + 1) 3399.9, 1739.5, 1712.5, 1629.6 PEX-12Y −488(M − 1) 3399.9, 1739.5, 1700, 1648.9 PEX-13Y − 487(M − 1) 3415.4,1739.5, 1708.6, 1658.5 PEX-14Y + 508(M + 1) 3390.3, 1739.5, 1700.0,1666.2, 1598.7, 1529.3, 1440.6, 756.0, 694.3 PEX-15Y − 522(M − 1)3390.3, 1739.5, 1716.4, 1648.9, 1602.6, 1538.9, 1444.4, 775.3, 690.4PEX-16Y + 483(M + 23) 3446.2, 1741.4, 1702.9, 1645.0 PEX-18Y + 531(M +23) 1741.4, 1708.6, 1643.1 PEX-21 + 469(M + 23) — PEX-21Y + 511(M + 23)3434.7, 1743.4, 1706.7, 1643.1 PEX-23Y + 517(M + 23) 3457.8, 1776.1,1712.5, 1639.2 PEX-24Y + 565(M + 23) 3434.7, 1737.6, 1708.6, 1643.1PEX-25Y + 521(M + 23) 3434.7, 1739.5, 1708.6, 1643.0, 1600.0, 1500.0PEX-26Y − 483(M − 1) 3457.8, 1776.1, 1712.5, 1639.2, 1600, 1469.5PEX-30Y + 525(M + 23) 3434.7, 1741.4, 1706.7, 1645.0 PEX-31Y − 513(M− 1) 3444.0, 1743.4, 1704.8, 1635.0 PAM-1Y — — 3430.8, 3203.2, 1743.4,1689.4, 1635.0 PAM-2Y — — 3426.9, 1706.7, 1685.5 PAM-5Y — — 3390.3,1712.5, 1654.7

TABLE 3 MS and ¹HNMR data of some compounds of the invention (¹HNMR dataof some compounds in Tab. 2 were omitted.) ESI-MS IonizationDeprotonated ¹HNMR δ (ppm), J (Hz), Solvent is CD₃Cl unless No. type ionpeak otherwise mentioned PEX-1 — — 0.94(d, 6H, 16.6), 1.70(s, 3H),1.95(d, 3H, 1.5), 3.88(dd, 2H, 1.8, 6.6), 5.93(d, 1H, 15.0), 6.55 (dd,1H, 15.0, 11.4), 7.25(m, 2H). PBX-1Y — — 0.94(d, 6H, 6.6), 1.60(s, 3H),1.98(s, 3H), 2.13 (s, 3H), 3.89(dd, 2H, 6.2, 1.8), 5.92(d, 1H, 15.0),6.56(dd, 1H, J=15.0, 11.7), 7.20(m, 1H), 7.27(d, 1H, 11.7). PBX-2Y +497(M + 23) — PBX-3Y — — 1.17(t, 3H, J=7.3), 1.60(s, 3H), 1.96(d, 3H,J=1.1), 2.41(q, 2H, J=8.1), 3.72(s, 3H), 5.92(d, 1H, J=15.0), 6.56(dd,1H, J=15.0, 11.7), 7.22(m, 1H), 7.26(d, 1H, J=11.7). PBX-4Y — — 1.60(s,3H), 1.96(s, 3H), 2.13(m, 3H), 3.86(t, 2H, 4.4), 4.27(m, 2H), 5.92(d,1H, 15.0), 6.55 (dd, 1H, 15.0, 11.7), 7.26(m, 2H). PBX-5Y + 488(M + 1) —PBX-6Y + 505(M + 23) — PBX-7Y + 551(M + 1) — PBX-8Y − 502(M − 1) —PBX-9Y + 511(M + 23) — PBX-10Y + 481(M + 23) — PBX-11Y + 513(M + 23) —PBX-12Y + 499(M + 23) — PBX-13Y — — 1.45(s, 3H), 1.83(d, 3H, 1.1),1.93(s, 3H), 3.21(t, 2H, 5.3), 3.53(t, 2H, 5.3), 5.81(d, 1H, 15.4),6.39(dd, 1H, 15.4, 11.4), 7.09(d, 1H, 11.4) PBX-14Y + 493(M + 1) —PBX-15Y + 472(M + 1) — PBX-16Y − 472(M − 1) — PBX-17Y + 559(M + 23) —PAM-7Y — — (in CD₃OD: 1.42(s, 3H), 1.76(s, 3H), 3.32(m, 4H), 5.93(m,2H), 6.37(dd, 1H, 14.9, 11.4), 7.06(d, 1H, 11.4) PAM-8Y — — 1.57(s, 3H),1.94(s, 3H), 2.11(s, 3H), 2.81(d, 3H, 4.1), 5.89(d, 1H, 15.1), 6.4(m,1H), 6.52 (dd, 1H, J=15.1, 11.6), 7.2(d, 1H, J=11.6).

Compounds of the invention with preferred antitumor actions have thefollowing structures:

No. R R₃ PEX-15Y methyl (m-hydroxyanilino) acyl PEX-25Y (α-furyl)methylene carboxyl PEX-31Y cyclohexylmethylene carboxyl

To evaluate the anti-tumor pharmacological activity of the compounds ofthe invention, the anti-proliferation activity thereof was assayed usingP388 murin leukemia cells and A549 human lung adenocarcinoma cells; theanti-angiogenic activity, a newer target of antitumor, was measuredusing human microvascular endothelial cells (HMEC). Pseudolarix acid-Band hydroxycamptothecin were used as reference compounds in theevaluation.

P388, A549 and HMEC Proliferation Assay

Materials: DMEM was purchased from Gibco (Life Technologies, GrandIsland, N.Y., USA). Sulforodamine B (SRB) and MTT were obtained fromSigma; TCA (acetic acid) and Tris base buffer were analytical pureproducts made in China.

SRB assay: Tumor (A549 and HMEC) cells in logarithmic growth were platedin DMEM media in the wells of a 96-well plate (5×10³ cells/90 μL/well)and serial dilutions of the compounds (10 μL) prepared in medium wereadded. Experiments at each concentration of compounds were performed intriplicate with controls of normal saline and cell-free withered wells.The plates were incubated at 37° C., 5% CO₂, for 72 hr. The media weredecanted and cells were fixed with cold (4° C.) 10% trichloroaceticacid, followed by incubation at 4° C. for 1 hr. Then plates were washedwith deionised water five times and allowed to air dry and stained byaddition of 100 μL/well SRB solution [0.4% SRB (Sigma) in 1% acetic acid(W/V)] for 15 min. Following staining, plates were quickly washed fivetimes with 1% acetic acid to remove unbound dye, and allowed to air dry.Bound dye was solubilized with Tris buffer (pH 10.5) prior to readingplates. The OD was measured with a multiwell spectrophotometer(VERSAmax, Molecular Devices, USA) at a wavelength of 520 nm.

MTT assay: P388 cells were seeded at a density of 1.5×10⁵/ml into wellsof 96-well plates and incubated with complete medium. After 24 hr,serial dilutions of the compounds (10 μL) prepared in medium were added.Experiments at each concentration of compounds were performed intriplicate with controls of cell-free withered wells and cell-freewithered wells at corresponding concentration of compounds, if it iscolored. The plates were incubated at 37° C., 5% CO₂, for 48 hr. Then,20 μL aliquots of MTT (Sigma) solution (5 mg/mL) in saline were addeddirectly to all the appropriate wells. The culture was continuouslyincubated for 4 hr. Then 50 μL “triplex solution” (10% SDS-5%isobutanol- 0.01M HCl) was added into each well. After the plates wereincubated at 37° C. overnight, the values of absorbance at 520 nm weremeasured by using a plate reader (VERSA Max, Molecular Devices).

The rate of inhibition on the growth of cancer cells was calculated bythe formula:Rate of growth inhibition=[1−(A _(520 treated) /A _(520 control))]×100%

The result was also expressed as IC₅₀ (the drug concentration reducingby 50% the absorbance in treated cells, with respect to untreated cells)that was calculated by Logit method. The mean IC₅₀ was determinedaccording to the data from three independent tests.

Preferred compounds of the invention screened by P388 model are asfollows, the structures thereof were shown in Table 1.

No. IC₅₀ (mol/L) No. IC₅₀ (mol/L) PEX-4Y 4.4 × 10⁻⁸  PEX-17Y   1.6 ×10⁻¹⁰ PEX-18Y 4.8 × 10⁻¹⁰ PEX-21Y <1.0 × 10⁻¹⁰ PEX-20Y 3.4 × 10⁻¹⁰PEX-25Y <1.0 × 10⁻¹⁰ PEX-24Y 2.6 × 10⁻¹⁰ PEX-31Y <1.0 × 10⁻¹⁰ PEX-30Y<1.0 × 10⁻¹⁰   Pseudolarix acid-B   8.3 × 10⁻¹⁰

Preferred compounds of the invention screened by A549 model are asfollows, the structures thereof were shown in Table 1.

No. IC₅₀ (mol/L) No. IC₅₀ (mol/L) PEX-14Y 9.8 × 10⁻⁸ PEX-15Y 15 × 10⁻⁸PEX-18Y 1.3 × 10⁻⁸ PEX-25Y 5.5 × 10⁻⁸ PEX-24Y 6.9 × 10⁻⁸ PEX-31Y <1.0 ×10⁻¹⁰  Pseudolarix acid-B 2.8 × 10⁻⁸

Preferred compounds of the invention screened by HMEC model, thestructures thereof were shown in Table 1.

No. IC₅₀ (mol/L) No. IC₅₀ (mol/L) PEX-8Y 9.6 × 10⁻⁷ PEX-25Y 1.0 × 10⁻⁹Pseudolarix acid-B 8.0 × 10⁻⁸ hydroxycamptothecin 4.7 × 10⁻⁷

The results of pharmacological screening has shown that the growthinhibition of many compounds of the invention on P388 and A549 cells wasbetter than that of pseudolarix acid-B. In HEMC model, the activities ofsome compounds were better than or equivalent to hydroxycamptothecin.The activities of several compounds exceeded that of pseudolarix acid-Bor hydroxycamptothecin in all of P388, A549 and HMEC models. Among them,some compounds are expectantly to become novel antitumor drugs.

Screening on Antifungal Activities

The action on Candida albicans was observed using liquid dilutingmethod. The drug concentration in the tube in which no growth of fungusobserved is the minimun inhibition concentration.

The action on Trichophyton rubrum was observed using agar dilutingmethod. The drug concentration in the Petridish on which no growth offungus observed is the minimun inhibition concentration. The specificdetermination was seen in the reference: Zhang Zuoran ed., <<Experimentof Medical Microorganism>>, Science Publishing House, 1998, pp.102-103.

Preferred antifungal compounds of formula (I) wherein R₁ is CO₂R′, R₂ isacetyl, R₃ is carboxyl, are as follows. Pseudolarix acid-B is areference compound.

MIC (μg/ml) MIC (μg/ml) (Candida (Trichophyton No. R′ albicans) rubrum)PEX-4Y ethyl 6.25 12.5 PEX-25Y (α-furyl)methylene 6.25 12.5 Pseudolarixacid-B 12.5 50.0

The results of the antifungal screen has shown that some of thecompounds of the invention have superior activities in comparison withpseudolarix acid-B and they are expected to become novel antifungaldrugs.

The invention will be further illustrated by the following examples, butthese examples are by no means intended to limit the invention.

EXAMPLE 1

Step 1:

0.060 g (0.139 mmol) of pseudolaric acid B and 10 mL of anhydrousethanol were added to a 25 mL round bottom flask. After the mixture wasstirred at room temperature, it was adjusted with sodium ethylate(prepared by the reaction of metal sodium and anhydrous ethanol) toabout pH 12. The reaction was then monitored by TLC till pseudolaricacid B disappeared. After the mixture was neutralized with acetic aciddropwise to pH=6, ethanol was removed on a rotatory evaporator. Theresidue was suspended in 10 mL water and then extracted with ethylacetate three times. The organic phases were combined and ethyl acetatewas removed on a rotatory evaporator, thus obtaining a crude product ofcompound PEX-4, which can be further purified by silica gel columnchromatography to obtain a pure product.

¹HNMR (CDCl₃) δ(ppm): 1.25 (t, 3H, J=7.13Hz), 1.56 (S, 3H), 1.91 (S,3H), 4.12 (m, 2H), 6.18 (d, 1H, J=15.11Hz), 6.52 (dd, 1H, J=15.11,11.24Hz), 7.13(m, 1H), 7.22(d, 1H, J=11.24Hz).

Compounds PEX-1, PEX-19 to PEX21 and PEX-30 were prepared in similarmethods.

Step 2:

10 mL of acetyl chloride was added to the reaction bottle with the abovecrude product. The reaction bottle was sealed and the mixture wasstirred with an electromagnetic stirrer. The reaction was monitored byTLC per hour till compound PEX-4 disappeared. The reaction was completedafter about 5 h, then exceeding acetyl chlorine was removed on arotatory evaporator and 10 mL water was added. The water phase wasextracted with ethyl acetate three times. The organic phases werecombined and evaporated to dry under reduced pressure. The residue wasloaded on a silica gel column (H60) and eluted with petroleum:ethylacetate:formic acid=3:1:0.1 to afford 0.0535 g of compound PEX-4Y, as awhite solid (yield 86.4%).

¹HNMR (CDCl₃) δ(ppm): 1.27(t, 3H, 7.23Hz), 1.59(S, 3H), 1.95(S, 3H),2.11(S, 3H), 4.15(m, 2H), 5.91(d, 1H, J=15.02Hz), 6.54(dd, 1H, J=15.02,11.42Hz), 7.18(m, 1H), 7.25(d, 1H, J=11.42Hz). IR(KBr) (cm⁻¹): 3434.7,1741.4, 1706.7, 1643.1.

Compounds PEX-1Y to PEX-31Y and compounds PBX-1Y to PBX-17Y wereprepared in similar methods.

EXAMPLE 2

0.432 g (1 mmol) of pseudolaric acid B was suspended in 40 ml ofdistilled water and 1.5 mmol aqueous methylamine (28%) was added at roomtemperature. Then 0.5 mmol acetic acid was added. The reaction was keptat ordinary temperature and pressure with stirring. The monitor of TLCshowed that the reaction completed after about 6 h. Then, the reactionmixture was diluted by 60 mL of water and extracted with ethyl acetatefive times. The organic phase was dried on anhydrous sodium sulfateovernight, and then filtered. The filtrate was evaporated to removeethyl acetate. The residue was subjected to a silica gel column andeluted with chloroform:methanol=15:1 to yield 0.400 g of PAM-8Y, as alight yellow solid (92.8%).

¹HNMR (CDCl₃) δ (ppm): 1.57(s, 3H), 1.94(s, 3H), 2.10(s, 3H), 2.81(d,3H, J=4.11Hz), 5.89(d, 1H, J=15.08Hz), 5.91(brs, 1H), 6.42(m, 1H),6.52(dd, 1H, J=15.08, 11.57Hz), 7.23(d, 1H, J=11.57Hz). IR (KBr) (cm⁻¹):3390.3, 1712.5, 1654.7, 1612.2.

Compounds PAM-1Y to PAM-8Y were prepared in similar methods.

EXAMPLE 3

0.05 g (0.116 mmol) of pseudolaric acid B and 3 mL cyclohexyl methanolof were added into a flask. To this mixture, powdered potassiumt-butoxide was added in batch with stirring at room temperature toadjust the mixture to pH 13. The flask was sealed and the mixture wasstirred for 24 h. The monitor of TLC showed that the reaction completed,then the reaction mixture was neutralized with anhydrous acetic acid topH=6. After the removal of cyclohexyl methanol and ethanol, the residuewas subjected to silica gel comlumn chromatography (petroleum:ethylacetate:formic acid=3:1:0.1) to afford 0.032 g of PEX-31Y, as a lightyellow solid (55.0%).

¹HNMR (CDCl₃) δ (ppm): 1.55(s, 3H), 1.94(s, 3H), 2.10(s, 3H), 3.89(m,2H), 5.89(d, 1H, J=15.01Hz), 6.52(dd, 1H, J=15.01, 11.35Hz), 7.19(m,1H), 7.26(d, 1H, J=11.35Hz). IR (KBr) (cm⁻¹): 3444, 1743.4, 1704.8,1635.0.

EXAMPLE 4

0.050 g (0.116 mmol) of pseudolaric acid B and 2 mL of dry α-furfurylmethanol were added into a 10 mL flask equipped with a reflux condenserand a calcium chloride tube, and the powder of potassium t-butoxide wasadded in batch with stirring at room temperature to adjust the mixtureto pH 11. The mixture was stirred for 3 h at room temperature, thenheated to 80-90° C. overnight. After being adjusted to pH 6 with aceticacid, the reaction solution was distilled under reduced pressure toremove furfuryl alcohol and ethanol. The residue was purified by silicagel column chromatography (petroleum:ethyl acetate:water=3:1:0.1) toobtain a brown solid, followed by RP-18 column chromatography(methanol:water=7:3) to afford 0.045 g of compound PEX-25Y, as a lightyellow solid (yield 78.1%).

¹HNMR (CDCl₃) δ(ppm): 1.59 (s, 3H), 1.91(s, 3H), 2.13 (s, 3H), 5.04 (d,1H, J=13.2Hz), 5.15 (d, 1H, J=13.2Hz), 5.91(d, 1H, J=14.8Hz), 6.38 (d,1H), 6.55 (dd, 1H, J=14.8, 11.6Hz), 7.21-7.27 (m, 4H). IR (KBr) (cm⁻¹):3434.7, 1739.5, 1708.6, 1643.1, 1600.0, 1500.0.

EXAMPLE 5

0.050 g (0.129 mmol), AgCN 0.172 g (1.28 mmol) and 0.5 ml ethyl etherwere added to a flask. At room temperature, 3 mLp-chlorine-phenyl-acetyl chlorine was dropped to the mixture. After 24h, the mixture was filtrated to remove AgCN and filtrate was thenremoved p-chlorine-phenyl-acetyl chlorine and ethyl ether underpressure. The residue was purified by silica gel column and eluted withpetroleum:ethyl acetate:formic acid=3:1:0.1 to give PEX-24Y 0.030 g(43.0%).

¹HNMR (CDCl₃) δ (ppm): 1.56 (s, 3H), 1.94 (s, 3H), 3.64 (s, 2H), 3.72(s, 3H), 5.87 (d, 1H, J=15.6Hz), 6.52 (dd, 1H, J=15.6, 11.2Hz),7.21-7.36 (m, 6H). IR (KBr) (cm⁻¹): 3434.7, 1737.6, 1708.6, 1643.1,1492.7, 1438.7, 810.0, 771.4.

EXAMPLE 6

Step 1: 0.030 g (0.069 mmol) pseudolaric acid B and 1 mL ethyl etherwere added and then 0.5 mL SOCl₂ was dropped at room temperature. Afterstirred for 1 h, the mixture was heated to 40-50° C. and kept at thetemperature for 3 h. The mixture was then cooled to room temperature andthe exceeding SOCl₂ was removed under pressure. 1 mL ethyl ether wasadded to form solution A.

Step 2: The flask containing 3 ml acetone, 1 mL water, 0.10 g (1.2 mmol)NaHCO₃ and 0.10 g (0.92 mmol) p-hydroxylanline was cooled to −2° C.Above solution A was slowly dropped and then the reaction was keep atabout 0° C. After about 1 h, the mixture was adjusted to pH=6 (aceticacid) and removed acetone under pressure. Water phase was extracted withethyl ether and the extract was purified by silica gel column(chloroform:methanol=10:1) to afford PEX-15Y 0.030 g (82.6%).

¹HNMR (DMCO) δ (ppm): 1.61 (s, 3H), 2.02 (s, 3H), 2.17 (s, 3H), 3.69 (s,3H), 6.12 (d, 1H, J=15.2Hz), 6.52-6.58 (m, 2H), 6.99 (d, 1H, J=11.0Hz),7.06-7.12 (m, 3H). IR (KBr) (cm⁻¹): 3390.3, 1739.5, 1716.4, 1648.9,1602.6, 1500.0, 1444.4, 775.3, 690.4.

EXAMPLE 7 Preparation of the Pharmaceutical Composition

Formula

Component Amount PEX-25Y 200 mg  Ethanol   2 ml Methylcellose 0.2 mlNormal saline 7.0 ml Tween 80 0.8 ml

Method of Preparation: 200 mg of PEX-25Y, 0.8 mg of Tween 80 and 2 mlethanol were added into a morta and ground homogeneously. 5 minuteslater, 7.0 ml of normal saline and 0.2 ml of methylcellose were added toform a solution containing 20 mg/ml PEX-25Y.

EXAMPLE 8

30 Kunming strain female mice weighing 18-22 g were used. Suspensions ofwell grown Sarcoma 180 cell were implanted subcutaneously into the rightaxilla region of the mice, about 4.5-5×10⁶ cells/mouse. The animals weregrouped randomly 24 hours after the implantation. Daily treatment withdrugs or normal saline commenced 1 day after implantation of S-180cells. Mice were administered by i.p. injection with vehicle orcompounds once a day for consecutive 6 days. All the mice wereeuthanized after the last administration. The mice and the tumors wereweighed. The average weights of tumors of each group were calculated andrate of inhibition of tumor growth in vivo was calculated using thefollowing formula:${{Growth}\quad{inhibition}\quad(\%)} = {\left( {1 - \frac{{Average}\quad{tumor}\quad{weigh}\quad{of}\quad{test}\quad{group}}{{Average}\quad{tumor}\quad{weigh}\quad{of}\quad{control}}} \right) \times 100\%}$

Results:

Number of mice Body weight (g) Tumor Group Dosage Beginning At lastBeginning At last weight (g) Inhibition % PEX-25Y  5 mg/kg 10 10 20.421.1 0.30 ± 0.23 64.3 PEX-25Y 10 mg/kg 10 9 20.4 19.7 0.26 ± 0.11 69.0PEX-25Y 15 mg/kg 10 10 20.5 21.2 0.28 ± 0.13 66.7 Control — 20 17 20.721.5 0.84 ± 0.25 —

The above examples are only used to illustrate the preparation ofcompounds and pharmaceutical compositions of the invention and thepharmacological experiment results thereof. Those skilled in the art canmake various modifications and alterations without departing from thespirit and scope of the invention. All these modifications andalterations will be covered by the claims.

1. Pseudolaric acid-B derivatives having general formula (I):

wherein (a) R₁ is cyano, heterocyclyl, COXR′ or CON(R″)₂, wherein X is Oor NH, R′ is H, cycloalkyl, alkyl, heterocyclic alkyl or arylalkyl, eachR″ is independently alkyl, cycloalkyl or heterocyclicalkyl; (b) R₂ is H,alkylacyl, arylalkylacyl, arylacyl or heterocyclylacyl; and (c) R₃ isCOXY, amino or halogen, wherein X is O or NH, Y is H, NH₂, hydroxy,alkyl, cycloalkyl, heterocyclicalkyl, hetroatom-substituted alkyl,tertiary amino-substituted ammonioalkyl, aryl, arylalkyl orpolyhydroxyalkyl, Provided that pseudolaric acid A, pseudolaric acid B,pseudolaric acid C, pseudolaric acid C₂, deacetoxyl pseudolaric acid A,deacetoxyl pseudolaric acid C₂, methyl pseudolarate A, deacetoxyl methylpseudolarate A, p-bromine phenacyl pseudolarate A, methyl pseudolarateB, p-bromine phenacyl pseudolarate B, methyl pseudolarate C, propylpseudolate C, iso-propyl pseudolate C, 19-demethoxyl iso-propylpseudolarate B, 19-demethoxyl iso-propyl pseudolarate C, 19-propyoxylpseudolarate B, 19-propoxyl pseudolaric acid C, 19-butoxyl pseudolaricacid B, and 19-butoxyl pseudolaric acid C are excluded.
 2. Pseudolaricacid-B derivatives according to claim 1 wherein R₁ is CONHR′ orCON(R″)₂, R₂ is H, an alkylacyl, an arylalkylacyl, an arylacyl, or aheterocyclic acyl, and R₃ is —COOH.
 3. Pseudolaric acid-B derivativesaccording to claim 1 wherein R₁ is —COOR′, R₂ is H, an alkylacyl, anarylalkylacyl, an arylacyl, or a heterocyclic acyl, and R₃ is —COOH. 4.Pseudolaric acid-B derivatives according to claim 1 wherein R₁ is—COOR′, R₂ is H, an alkylacyl, an arylalkylacyl, an arylacyl, or aheterocyclic acyl, and R₃ is COXY.
 5. A process for preparing thepseudolaric acid-B derivatives according to claim 1, comprising thesteps of: a. In case R₁ is CONHR or CONR′R″, aminolyzing a compound offormula (I) wherein R₁ is COOR′ in a water solution of an amine (saidcompound:said amine=1:1˜1:300) in the presence of an acid catalyst atthe conditions of temperature from −10° C. to 100° C. and pH 1-6; b. Incase R₁ is COOR′, alcoholyzing a compound of formula (I) wherein R₁ isCOOR′ with an excess alcohol (said compound:said alcohol=1:1˜1:500) inthe presence of an alkali catalyst under anhydrous condition at atemperature from 0° C. to the reflux temperature of the solvent and pH9-14; c. In case R₂ is alkylacyl, aryl-substituted alkylacyl, arylacylor heterocyclylacyl, acylating a compound of formula (I) wherein R₂ is H(said compound:acylating agent=1:5˜1:500) at a temperature from 0° C. to80° C.; or d. In case R₃ is COXY, reacting a compound of formula (I)wherein R₃=COOH with an excess acyl halogenating agent to form an acylhalide, then reacting said acyl halide with an alcohol or an amine inthe presence of an acid scavenger at a temperature between −20˜30° C. 6.The process according to claim 5, wherein said acid catalyst is selectedfrom a group consisting of HCl, CH₃CO₂H, H₂SO₄ and H₃PO₄.
 7. The processaccording to claim 5, wherein said alkaline catalyst is selected from agroup consisting of sodium amide, potassium amide, sodium alkoxide andpotassium alkoxide, preferably, sodium alkoxide and potassium alkoxide.8. The process according to claim 7, wherein said alkaline catalyst isselected from sodium alkoxide and potassium alkoxide.
 9. The processaccording to claim 5, wherein said acylating agent is an acyl halide.10. The process according to claim 5, wherein said halogenating agent isselected from a group consisting of SOCl₂, POCl₃, PCl₅, S₂Cl₂, PBr₃ andPCl₃.
 11. The process according to claim 5, wherein said acid scavengeris selected from a group consisting of organic amine, CaCO₃, K₂CO₃,Na₂CO₃, NaHCO₃, KOH and NaOH.
 12. A pharmaceutical composition for thetreatment of tumors or inhibition of fungi, comprising a therapeuticallyeffective amount of compound of claim 1 and a pharmaceuticallyacceptable carrier.